Targeting of mutant-p53 and MYC as a novel strategy to inhibit oncogenic SPAG5 activity in triple negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient’s outcome, and reliable biomarkers for effective anticancer therapies.


INTRODUCTION
Breast cancer (BC) is the most frequent cancer among women worldwide.Current histopathological classification and targeted treatment significantly improved prognosis of luminal and HER-2 enriched tumors, whereas triple negative breast cancer (TNBC), routinely classified as a group of highly heterogenous disease, still lacks of specific prognostic and therapeutic biomarkers [1].Beyond transcriptomic classification of TNBC into Basal-like 1/2, mesenchymal, luminal and androgen receptor positive subtype, further stratification is critical to dissect their genetical landscape and plan effective anticancer therapy [2][3][4].
Sperm associated Antigen 5 (SPAG5) is an essential component of mitotic spindle, needed for chromosome alignment and sister chromatids segregation during anaphase [5][6][7].Frequently overexpressed in advanced BC [8], including TNBC, SPAG5 is considered to be an efficient prognostic factor [9,10].Amplification of SPAG5 gene is associated with BC growth, positive lymph node status, anthracycline vulnerability, poor prognosis, and TP53 mutations [11].TP53 is the most common mutated gene across cancers, including BC. TP53 gene, encode for a transcription factor involved in cell cycle regulation, apoptosis, DNA repair, and metabolic processes [12].
In BC, TP53 mutations account for about 30% but their frequency markedly increases to ~80% in TNBC [6].Major alterations in the TP53 gene are missense mutations, which are frequently located in the DNA binding domain (R175, R248, R249, R273, R282), and result in gain of oncogenic activity [13].In cancer cells, the oncogenic function of mutant p53 proteins (mut-p53) can be enhanced through interaction with other transcription factors or coactivator, including the Hippo pathway transducer YAP [14,15].
Previously, we showed that SPAG5 depletion strongly impaired proliferation and migration of TNBC cell lines, and identified SPAG5 as a direct transcriptional target of YAP/TAZ/ TEAD [16].Here, we analyzed the expression levels of SPAG5, YAP and p53 transcripts in METABRIC dataset, and found that the expression levels of YAP and SPAG5 increased along with p53 mutations in more aggressive BC histotypes.This, was associated with elevated level of TP53 mutant-dependent MYC signature.MYC depletion, or inhibition by small-molecule inhibitors, reduced SPAG5 expression, clonogenicity, and sensitized TNBC cells lines to cisplatin, 5-fluorouracil and paclitaxel.We further found that SPAG5 is a direct target of MYC, and collectively showed that mut-p53, YAP and MYC coordinately function to regulate SPAG5 expression.Our data, introduce mut-p53-MYC-SPAG5 expression as bona fide predictors of patient's outcome, and reliable biomarkers for effective anticancer therapies.
Bright-field imaging of organoids was performed on an NEXCOPE microscope.

Colony-formation assay
BC cell lines were grown in 60-80% confluence and transfected with siRNAs using Lipofectamine RNAi MAX, according to the manufacturer's instructions, or treated with the indicated compounds.After 48 h, 500 cells were seeded in six-well dishes (Corning-Costar, Tewksbury, MA, USA).Fresh media (25%) was added every 3 days.After 7-10 days of culture, the colonies were stained with crystal violet and counted.

Cell viability assay
In all, 8 × 10 2 cells were seeded into 96-well plates.Cell viability was assessed using ATPlite assay (Revvity, Massachusetts, USA), according to the manufacturer's instructions.Combination Index (CI) was calculated by calcusyn software according to the manufacturer's instructions.

PDTO morphological measurement
The measurement of the perimeter, width, length, and area dimensions of organoids was assessed using the Opera Phenix® Plus high-throughput microplate confocal imager (Revvity, Massachusetts, USA) and calculated using Harmony High-Content Imaging and Analysis Software [19].

Promoter analysis
FASTA sequences of human SPAG5 (NM 06461) promoter (5000 bp upstream of the TSS) were downloaded from UCSC Genome Browser on-Fig. 1 Expression levels of SPAG5, YAP and mutant P53 and their correlation to a p53-dependent MYC signature in breast cancer patients.A, B Heatmaps (on the left) of normalized expression levels of SPAG5, YAP and p53 status in METABRIC dataset.Pie-charts (in the middle) frequencies of p53 mutations in breast cancer patients from METABRIC dataset stratified for combined expression of SPAG5 and YAP.Tables (on the right) breast cancer histotype classification in the different group of breast cancer patients stratified for combined expression of TP53, SPAG5 and YAP.C Box-plot of SPAG5 expression in MDA-MB-468 breast cancer cells 48 h post sip53, siYAP or siCTRL transfection.siRNAs transfection assessed by RNA sequencing analysis.Volcano plots of all modulated transcripts as assessed by RNA sequencing of sip53 (D) or siYAP (E) transfected cells.Red dots mark upregulated and green dots downregulated genes.F Box-plot of expression levels of TP53-MYCtarget genes signature in breast cancer patients from METABRIC dataset, stratified for different combined levels of SPAG5 and YAP expression signatures.G Pie-charts represent the frequencies of TP53-MYC-target genes signature expression in breast cancer patients (left panel) and in TNBC patients (right panel) from METABRIC dataset, stratified for different combined levels of SPAG5 and YAP signatures.Heatmaps of normalized expression levels of SPAG5, YAP, TP53-MYC-target genes signature, and p53 in breast cancer patients (H) and in TNBC patients (I) from METABRIC dataset.line database.LASAGNA-Search 2.0 were used to identify predicted transcription factor binding sites.TRANSFAC matrices were used for the analysis.

ChIP experiments
Chromatin immunoprecipitation was performed as previously reported [16].The chromatin solution was immunoprecipitated with rabbit monoclonal anti-MYC-p(Ser62) (Cell Signaling) or rabbit polyclonal anti-H4Ac (2594, Cell Signaling).The immunoprecipitations were performed using Pierce ChIP-grade Protein G magnetic beads (Thermo Fisher Scientific).The immunoprecipitated and purified chromatin was subjected to RT-qPCR.The promoter occupancy was analyzed by RT-qPCR using the SYBR Green assay (Applied Biosystems).Normalization was performed to the amount of input chromatin.

Statistical analysis
Normalized gene expression of BC patients was obtained from Metabric dataset and Broad Institute TCGA Genome Data Analysis Center (2016); TCGA data from Broad GDAC Firehose 2016_01_28 run.Broad Institute of MIT and Harvard.Dataset.https://doi.org/10.7908/C11G0KM9.
DEseq2 pipeline was used for pre-process analysis.
Unsupervised hierarchical clustering was performed to identify specific patterns of gene expression using the Euclidean distance metric and average linkage.
Survival and progression-free survival analyses were conducted using the Kaplan-Meier method.To determine the statistical significance of differences between survival curves, a log-rank test was applied.High and low expression values for subgroups of patients were assessed by calculating z-scores of a single gene or by the z-scores of the mean value of a signature of genes.
The impact of clinical variables on survival curves was investigated using a multivariate Cox proportional hazard regression model.
Statistical significance for the modulation of a single gene among patient subgroups was inferred using the Student's t-test when comparing two groups or the ANOVA test when comparing more than two groups.

Pathway analysis
A Gene Set Enrichment Analysis (GSEA) was conducted using the GSEA software available at https://www.gsea-msigdb.org/gsea/index.jsp.This analysis utilized curated gene sets from the Molecular Signature Database (MSigDB) derived from KEGG, Hallmark, and Reactome collections.The GSEA was run in preranked mode using the classic metric, and 1000 permutations were performed to assess the statistical significance of pathway enrichment.Genes were ranked based on the formula SCORE = sign (FC) * (-log10Pvalue), which combined fold change (FC) and statistical significance (P-value) information.The above analyses were performed using MATLAB R2022a software.

Expression levels of SPAG5, YAP and mut-p53 in BC subtypes
Recently, we showed that in BC SPAG5 expression is transcriptionally sustained by YAP-TAZ-TEAD interaction [16], while previous reports suggest that mut-p53 proteins frequently crosstalk with YAP or other determinants of the Hippo signalling pathway [14].We therefore investigated whether YAP, mut-p53 and SPAG5 were mutually aberrantly expressed in BCs patients.
We classified 1969 BC patients from METABRIC dataset, containing clinical and genomic data from primary BC, into four different groups, based on the expression levels of SPAG5 and YAP signatures: SPAG5 high /YAP high , SPAG5 low /YAP low , SPAG5 high / YAP low , SPAG5 low /YAP high , (Heatmap Fig. 1A, B, Fig. S1A, B).For each group, we analysed TP53 status wild-type (wt-TP53) or mutated, and the prevalence of BCs histotypes (Normal-like, Basal-like, HER-2, luminal-A, luminal-B) as a measurement of tumor malignancies (Pie-charts Fig. 1A, B, Fig. S1A, B). Figure 1A depicts patients characterized by SPAG5 high and YAP high : in this group higher frequency of mut-TP53 (62.4%) was associated with a higher risk to develop more aggressive tumor histotype (Basal-like = 58%).In contrast, patients carrying wt-p53 protein with SPAG5 high and YAP high , mainly developed luminal features,  while the frequency of Basal-like histotype decreased to 16% (Table in Fig. 1A).Opposite scenario was observed by analysing patients with SPAG5 low and YAP low .In this group, TP53 mutations were less prevalent compared to the other categories (14.7%) and the incidence of basal-like subtype was decreased to 6% (Fig. 1B).We also considered patients with SPAG5 low / YAP high or SPAG5 high /YAP low .Beyond similar frequencies of TP53 mutations 28.5% in SPAG5 high /YAP low and 33% in SPAG5 low / YAP high groups, 41% of patients with YAP high and mut-p53 were associated with basal-like subtype (Fig. 1SA), while SPAG5 overexpression act as a self-sufficient supporter of tumor growth.Of interest, normal-like phenotype shows the lower prevalence when SPAG5 is highly expressed (Fig. 1SB).
Collectively, these observations suggest that in BC patients, SPAG5, YAP and mut-p53 were concomitantly expressed and clinically associated with tumor aggressiveness.

YAP and mut-p53 regulate SPAG5 expression independently
Previous studies have shown that mut-p53 and YAP recruit transcription factors to the promoters of shared target genes, supporting cell cycle progression and tumor growth [14,20].RNA-sequencing of MDA-MB-468 TNBC cells depleted of mutant TP53 (R273H) or YAP genes, showed reduced SPAG5 mRNA expression in these cells (Fig. 1D, E), suggesting that both YAP and mut-p53 regulate SPAG5 expression.Consistent with earlier reports [20], GSEA and KEGG pathway analysis identified cell cycle and mitotic checkpoint pathways regulated by mut-p53 and YAP.Among the negatively enriched pathways in TP53-depleted cells, we focus on MYC targets, because of its central oncogenic signal in BC and its compelling role in anticancer target therapy (Fig. S1C).Furthermore, concomitant high expression of mut-p53 and MYC is associated with poor prognosis in TNBC patients [21][22][23][24].
SPAG5, mut-p53, YAP and TP53-MYC-target gene signature are prognostically relevant for TNBC Normalized high expression of SPAG5 and YAP were significantly associated with high levels of a TP53-dependent MYCtarget gene signature (TP53-MYC target signature) in all BC (p = 5.1277 e-77 ) and in TNBC patients (p = 0.001) (Figs.1F, S1E) [24].Notably, 75% of all BC and 65% of TNBC patients with SPAG5 high and YAP high were also highly enriched in TP53-MYC target signature (Fig. 1G).Furthermore, TP53 mutations prevailed within patients with high levels of SPAG5, YAP and TP53-MYC signature (Fig. 1H, I).COX multivariate analysis adjusted for tumoral stage, nodal stage, histotype, age and menopausal state, further predicts that SPAG5, mut-p53 and YAP are prognostic determinants for BC, independently or in association with TP53-MYC signature (Table 1).Collectively, these findings reveal that expression of SPAG5, YAP, mut-p53 and TP53-MYC signature, are mutually connected and significantly prognostic for BC patients.

c-MYC depletion reduces SPAG5 expression and clonogenic ability of TNBC cell lines
To further explore the relation between SPAG5 and MYC, we assessed their association in METABRIC dataset.As shown, high levels of SPAG5 and YAP were significantly associated with c-MYC expression in BCs patients (p = 8.0932 e-23) (Fig. 3A).A similar trend was observed in TNBC patients, although it was not statistically significant (p = 0.12) (Fig. S3A).Therefore, we knocked down c-MYC in MDA-MB-468, MDA-MB-231 and SUM-159 TNBC cell lines.SPAG5 mRNA (Fig. 3B, E, H) and protein (Fig. 3C, F, I) levels were markedly reduced in MYC-depleted cells.Expression of NME1 G3BP1, XRCC6, CCT3 and TCP1 [24], were decreased (Fig. S3B), and the number of the colonies formed by TNBC cell line were also significantly reduced (Fig. 3D, G, J).These results, together with the downregulation of SPAG5 and cyclin-B1, and upregulation of CDK inhibitor p21 proteins, suggest that depletion of c-MYC reduced SPAG5 expression and consequently BC cell proliferation (Fig. 3C, F, I).To corroborate these findings, we used the small molecule MYC-inhibitor, MYCi975 [25].Noticeably, MYCi975 induced a dose dependent downregulation of SPAG5 mRNA and protein levels,  SPAG5 is a direct transcriptional target of MYC Two different c-MYC binding sites were predicted by Lasagna 2.0 algorithm on SPAG5 promoter (Fig. 3Q).ChIP-PCR assay demonstrated a significant enrichment of active MYC-p(Ser62) on the selected regions of SPAG5 promoter (Fig. 3R, T), concomitantly with increased Histone H4 acetylation, marker of transcriptional active chromatin (Fig. 3S, U).Furthermore, p53 or YAP depletion (Fig. S3H), markedly reduced MYC-p(Ser62) recruitment on SPAG5 promoter and histone acetylation on the selected regulatory regions (Fig. 3R-U).SF3B3, MYC-target gene, was used as a positive control (Fig. S3J) [24].Collectively these findings show that SPAG5 is a direct transcriptional target of MYC, p53 and YAP axis.
BET-inhibitor sensitizes TNBC cell to chemotherapy SPAG5 knockdown sensitized TNBC and cervical cancer cell lines to Olaparib and Taxol [9,33].Therefore, we tested whether JQ-1 treatment, which reduces SPAG5 expression, synergies with cisplatin to impair proliferation of TNBC cells.
Cisplatin, 5-Fluorouracil, and Paclitaxel slightly inhibited colony formation of TNBC cells, when administer as a single agent (Figs.5H-J, S5E-G, S5J).However, combination of cisplatin, paclitaxel, or 5-Fluorouracil with JQ-1 farther reduce clonogenic ability of MDA MB-231, compared with single agent treatments (Fig. 5H, S5J).These results suggest that the synergistic effect of these compounds is partially mediated by impairment of MYC activity, and consequently of SPAG5 expression.
inhibitors reduced SPAG5 expression via MYC, and sensitized TNBC cells to conventional therapy.To determine whether Dasatinib can synergize with cisplatin to inhibit SPAG5 expression and TNBC cell proliferation, we calculated their combination index in MDA-MB-231 and MDA-MB-468 cell lines (Fig. 5Q, R).As shown, pretreatment with Dasatinib sensitized MDA-MB-231 (Table 2C) and MDA-MB-468 (Table 2D) to cisplatin treatment (Fig. 5Q, R).As shown in Fig. 5 BET inhibitors and DASATINIB sensitized TNBC cell lines to chemotherapy.A-C Box-plot of SPAG5 expression in Responder and Non-Responder TNBC patients to 5-FU/DOXORUBICIN/CITOXAN (A) or TAXANE (C) treatments.B-D Roc Curves of SPAG5 expression in predicting Responder and Non-Responder TNBC patients to 5-FU/DOXORUBICIN/CITOXAN (B) or TAXANE (D) treatments.E, F Viability Assay.Synergy between JQ-1 and cisplatin was evaluated by cell viability assay using the ATPlite kit in MDA-MB231 (E) and in MDA-MB 468 (F) cells.G Western blot analysis of SPAG5, MYC-p(Ser62), c-MYC, and p21 protein levels in whole-cell lysate of MDA-MB231 cells, after treatment for 72 hr with 125 nM JQ-1 or 1 μM CDDP.Clonogenic assay.Representative micrographs of colonies formed by MDA-MB-231 (H), MDA-MB468 (I), and SUM-159 (J) cell lines pre-treated for 72 hr with 62.5 nM of JQ-1, and 1 μM of cisplatin before seeding at clonal density.After 7-10 days later, colonies were stained with crystal violet and counted.Column graphs show colony count and P value from three independent experiments.Western Blot analysis of SPAG5, MYC-p(Ser62), c-MYC, and p21 protein levels in whole-cell lysate of MDA-MB-231 cell line after treatment for 72 hr with 1 μM of CPI-0610, and 1 μM of cisplatin (K), 0.1 nM Paclitaxel (M) and 2.5 μM of 5-Fluorouracil (O).Clonogenic assay.Representative micrographs of colonies formed by MDA-MB-231 cell line pre-treated for 72 h with 0.5 μM of CPI-0610 and 1 μM of cisplatin (L) 0.1 nM Paclitaxel (N) and 2.5 μM of 5-Fluorouracil (P), before seeding at clonal density.After 7-10 days later, colonies were stained with crystal violet and counted.Column graphs show colony count and P value from three independent experiments.Q, R Viability assay.Dasatinib synergizes with cisplatin in MDA-MB231 (Q) and MDA-MB 468 (R) cells as evaluated by cell viability assay using the ATPlite kit.S Western blot analysis of SPAG5, MYC-p(Ser62), p21 and cleaved PARP protein levels in MDA-MB-231 cells following treatment with 500 nM DASATINIB and 0.5 μM of cisplatin.T Clonogenic assay.Representative micrographs of colonies of MDA-MB-231 cells pre-treated for 48 h with 25 nM DASATINIB and 0.3 μM cisplatin before seeding at clonal density.After 7-10 days later, colonies were stained with crystal violet and counted.Column graphs show colony count and P value from three independent experiments.(*P value < 0.05; **P value < 0.001).were obtained in response to the combined treatments (Fig. 5T).Collectively, these findings suggest that in preclinical models, undirected pharmacological targeting of MYC-SPAG5 axis perturb essential oncogenic features of TNBC.

Targeting of SPAG5 expression reduces viability of patients derived tumor organoid (PDO)
To translate our findings on three-dimensional (3D) system we used TNBC patients derived tumor organoids (TNBC-PDTOs) [35][36][37].RNA-seq analysis reveals similar expression levels of SPAG5, MYC, TP53, YAP1, G3BP1, XRCC6, CCT3, NME1 genes between parental tumoral tissues and TNBC-PDTOs (p = 0.0002, R = 0.9560 and p = 0.0035 and R = 0.8848) (Fig. 6A, B Table 3).Indeed, we analyzed organoids viability following JQ-1, CPI-0610 or Dasatinib treatments, alone or in combination with cisplatin.As shown in Fig. 6C, D when administrated as a single agent treatment, cisplatin slightly reduced viability of TNBC-PDTO.JQ-1 treatment inhibited TNBC-PDTO proliferation by almost 30%, while combination of JQ-1 with cisplatin inhibited TNBC-PDTO viability by almost 60%, highlighting the potency of this combination.We also tested the efficacy of CPI-0610.CPI-0610 alone, reduced the viability of TNBC-PDTO, but more profoundly together with cisplatin (Fig. 6B, D).Dasatinib had strong effect on PDTO viability as a single drug, and even stronger effect in combination with cisplatin (Fig. 6B, D).Therapeutic interventions can modify organoid morphology.To assess the efficacy of drug treatments on TNBC organoids, we measured perimeter, length, width, and area.As shown in Fig. 6E, F CDDP treatment resulted in minimal changes in these parameters, whereas JQ1, Dasatinib and CPI-0610 had more pronounced effects on organoid architecture.Furthermore, the combination of JQ1, CPI-0610 and Dasatinib with cisplatin led to more significantly reduced the perimeter, length, width, and organoids area, corroborating the viability data (Fig. 6E, F).Altogether, our results, marked SPAG5 expression as a bona fide biomarker to identify patient's responder and/or non responders to MYC inhibitors and their combination with chemotherapy.

DISCUSSION
TNBCs are high invasive and proliferative tumors, which hide extensive inter-and intra-tumoral heterogeneity [2,38].Due to the lack of effective target, the current therapeutic approach for TNBC is cytotoxic chemotherapy, but acquired resistance and relapse are hurdle still difficult to overcome [39].In our study, we used gene expression profiling and mechanistic assays to uncover a complex oncogenic network regulating SPAG5 expression in TNBC.
SPAG5 promotes proliferation and chemoresistance, inhibits apoptosis and its overexpression is associated with poor prognosis in many different tumors [10,40].Frequently high expressed in recurrent and metastatic BCs, in TNBC, elevated SPAG5 expression was associated with high levels of Ki67, TOP2A, BIRC5, AURKA, BUB1, and ATR-BRCA pathways [8,9,41,42].The large expression of cell cycle related genes increased immune tumoral infiltration; the positive association between cancer's antigens and infiltrating immune cells is one of the suggested criteria applied to develop effective anti-cancer vaccine.Interestingly, SPAG5 overexpression was correlated with high levels of lymphocyte CD8+, macrophages, neutrophils, dendritic and B cells, positively associated with PD-1/PDL1, LAG3, GZMB and CTLA4 and SPAG5 antigens were suggested as valid immune stimulatory targets for anticancers therapy [40,43,44].
Over 80% of TNBC express mut-p53 protein [45].Previous data links SPAG5 overexpression with p53 signaling in HCC and lung cancer [40,46].Here, we demonstrated that in BC cell lines depletion of mut-p53 reduce SPAG5 transcripts and proteins expression.Moreover, mut-p53 directly recruits transcription factors on SPAG5 promoter.Accordingly, targeting of mut-p53 impairs c-MYC activity and SPAG5 expression.It has been reported that in TNBC, by interacting with MYC-BP, SPAG5 promotes MYC transcriptional activation [9].Here we demonstrated that SPAG5 is a direct transcriptional target of MYC, raising the possibility that BC progression was partially sustained by a MYC-SPAG5 positive feedback loop.Some pre-clinical studies, evidenced antitumoral activity and MYC sensitivity to BET-inhibitors and Dasatinib; nevertheless, their limited clinical application was bounded by the lacks of specific and sensitive biomarkers [47].We show that in MDA-MB-231 TNBC cell line, JQ-1 and Dasatinib directly impaired MYC-p(Ser62) recruitment and Histone H4 acetylation on SPAG5 promoter, reduced SPAG5 expression and oncogenic proprieties of TNBC cell lines.We tested SPAG5 expression as an effective predictive biomarker for advanced BET-inhibitors compounds: CPI-0610, INCB054329 and OTX015 treatments, markedly reduced MYCp(Ser62) activity and SPAG5 expression, leading TNBC cells to growth arrest.
Our translational approach provides strong evidence that genetic profiling of TNBC should improve selection of potential actionable targets, that is one of the primary issues in the development of advanced precision medicine.Along with chemotherapy, novel anticancer strategies are mandatory to improve TNBC patient's prognosis, and, in this context, SPAG5 overexpression should be used as a predictive biomarker for effective anticancer therapies (Fig. 6C).

Fig. 3
Fig. 3 MYC directly binds SPAG5 promoter.A, B Box-plots expression levels of c-MYC in breast cancer patients (A) from METABRIC dataset, stratified for different combined levels of SPAG5 and YAP signature expression.qPCR analysis of SPAG5 expression levels in SUM-159 (B), MDA-MB-231 (E) and MDA-MB-468 (H) 48 h post transfection with siGFP and siMYC.Western blot analysis shows the protein levels of SPAG5, c-MYC, cyclin B1 and p21 protein levels of whole-cell lysate of SUM-159 (C), MDA-MB-231 (F) and MDA-MB-468 (I) 48 hr post transfection with siGFP and siMYC.D, G, H Clonogenic assay.Representative micrographs of colonies formed by SUM-159 (D), MDA-MB-231 (G) and MDA-MB-468 (J) cells transfected with siGFP and siMYC.After 7-10 days, colonies were stained with crystal violet and counted.Column graphs show colonies number and P value from three independent experiments.K-N qPCR analysis of SPAG5 expression level in MDA-MB-468 (K) and MDA-MB-231 (N) assessed by quantitative PCR 48 hr post treatment with 2.5 μM −5 μM −7.5 μM of MYCi975.L-O Western blot analysis of SPAG5 and p-MYC protein levels in whole-cell lysate of MDA-MB-468 (L) and MDA-MB-231 (O) cells 48 hr post treatment with 2.5 μM −5 μM −7.5 μM of MYCi975.M-P Clonogenic assay.Representative micrographs of colonies formed by MDA-MB-468 (M), MDA-MB-231 (P) treated for 48 hr with 2.5 μM −5 μM −7.5 μM of MYCi975, before seeding at clonal density.After 7-10 days, colonies were stained with crystal violet and counted.Column graphs show colony count and P value from three independent experiments.Q Schematic representation of the SPAG5 promoter with the putative MYC-binding sites as predicted by LASAGNA.Search 2.0.R-T ChIP analysis of the MYC-p(Ser62) binding on SPAG5 promoter in MDA-MB-468 cells after TP53 and YAP interference, detected by RT-qPCR analysis.S-U Transcriptional active chromatin evidenced by anti-H4-Acetylate antibody.Data shown as one of three independent replicates.(*P value < 0.05; **P value < 0.001).
A recent phase II clinical trial documented the safety of combined treatment of Dasatinib, plus Trastuzumab and Paclitaxel in HER-2-positive metastatic BC patients with acquired resistance to Trastuzumab (NCT01306942, EudraCT 2010-023304-27) [34].

Fig
Fig. 5S, cisplatin or Dasatinib treatment alone, had no or little effect, respectively, on SPAG5 expression but their combination markedly reduced SPAG5 level in MDA-MB-231 cells.Furthermore, a significant reduction of MDA-MB-231 colonies

Table 1 .
COX multivariate analysis adjusted for T, N, stage and histotype, age and menopausal state, of SPAG5, mutant p53, YAP and TP53-MYC signature expression in breast cancer patients from METABRIC dataset.